[摘要]目的构建并筛选携带针对Epstein-Barr病毒(EBV)潜伏膜蛋白基因LMP1的pSUPER retro RNAi逆转录病毒载体以及稳定产毒的细胞克隆。方法 用DNA重组技术，将60 bp能转录产生靶向LMP1小发夹RNA(shRNA)的寡核苷酸序列，定向克隆入逆转录病毒载体pSUPER retro，脂质体法将重组逆转录病毒载体转染包装细胞系PA317，G418筛选建立稳定产生逆转录病毒的细胞克隆。结果重组逆转录病毒载体经限制性 内切酶酶切，电泳后可观察到7167bp和281 bp两条DNA条带;测序鉴定结果表明序列正确;重组载体转染包装细胞。可表达绿色荧光蛋白，经G418筛选，得到抗性细胞克隆。结论 特异性沉默EBV潜伏期基因LMP1的pSUPER retro RNAi逆转录病毒载体以及稳定产毒的细胞系构建和筛选成功。

    [关键词]shRNA;LMP1;PA317;逆转录病毒载体;Epstein-Barr病毒

    CONSTRUCTION OF pSUPER RETRO RNAi SYSTEM OF THE SPECIFIC SILENCING EBV LATENT MEMBRANE GENE LMP1

    YIN FAN， WANG XIAO-FENG， LUO BING

    (Department of Microbiology， Qingdao University Medical College，Qingdao 266021，China) [ABSTRACT] Objective To construct and identify a recombinant retroviral vector pSUPER-LMP1 that target EBV latent membrane protein gene 1 (LMP1) and a stable virus-producing cell line. Methods The 60 bp encoded targeting LMP1 gene shRNA sequence was cloned into a retroviral vector pSUPER retro with DNA recombinant technique. The recombinant vector was identified by the electrophoresis analysis of restriction enzyme digestion and DNA sequencing. The packaging cell PA317 was trans-fected with this recombinant plasmid using liposome-based transfection and the stable intergrant was selected by using G418 me- dium. Results The electrophoresis of EcoRⅠand HindⅢ digested products showed two DNA fragments ......